Advances in Applied and Pharmaceutical Sciences Journal TM Volume, Issue 3 (27), 5-54 Available online at www.aapsj.in Research Article Stability Indicating In-Vitro Dissolution Testing of Gemifloxacin and Ambroxol In Pharmaceutical Dosage Forms Zahid Zaheer*, Mirza Shahed Baig, Qazi Yasar, Wasim Ahamad,, Md. Imran Anees, Altamash Ansari, Neha Quadri Department of Quality Assurance, Y.B Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza Bagh, Aurangabad 43, (M.S),India Article information Received: November 27 Received in revised form: 22 November 27 Accepted: 2 December 27 Available online: January 28 Subject: Pharmaceutical Sciences Branch: Pharmaceutical Analysis *Corresponding author: Dr. Zahid Zaheer Department of Quality Assurance, Y.B Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza Bagh, Aurangabad 43, (M.S), India Email: zaheerzahid@gmail.com DOI:.26768/AAPSJ..3.5-54 Quick Response Code tract A simple, specific, precise and accurate reverse phase liquid chromatographic method has been developed and validated Stability indicating dissolution media for the determination of Gemifloxacin (GEM) (fluroquinolone antibiotic) and Ambroxol (AMB) (mucolytic) in pharmaceutical tablet dosage forms was performed and the stability of Gemifl oxacin and Ambroxol was tested in various dissolution media such as.m HCl, ph.2 KCl-HCl buffer and ph 5.8, 6.2, 6.6, 7., 7.4 and 7.8 phosphate buffers separately. The stability was tested at room temperature and 37oC (incubator) for, 24, 48 hrs. The samples were studied for stability and which samples show stability (least % CV) was selected for further study. Stability studies of GEM and AMB show that drug is stable in.m HCL, ph 6.6 and ph 7. phosphate buffers in the UV spectra for a period of 48 hrs. New First order derivative spectroscopic method was developed for dissolution studies and λmax for GEM was found to be 245nm (zero crossing of AMB) and λmax of AMB was found to be 292nm (zero crossing of GEM) in.m HCl, ph 6.6 and ph 7. phosphate buffers respectively. Calibration curve was plotted in the above three media and linearity of the calibration curve of GEM were found to be -5 µg/ml for AMB 2- µg/ml. Dissolution profiles of GEM and AMB in selected dissolution media such as.m HCL, ph 6.6 and ph 7. phosphate buffers was carried out successfully. The method was validated as per ICH guidelines. Keywords: Gemifloxacin, Ambroxol, Stability indicating, Dissolution media, First order derivative method. Cite this article Z. Zaheer, MS Baig, Q Yasar, W Ahamad,, MI Anees, A Ansari, N Quadri, Stability Indicating In-Vitro Dissolution Testing of Gemifloxacin and Ambroxol In Pharmaceutical Dosage Forms Advances in Applied and Pharmaceutical Sciences Journal 27, (3);5-54 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4./). 27 Advances in Applied and Pharmaceutical Sciences Journal (AAPSJ) e-issn No 2457-842, [Available online at aapsj.in] 5
Introduction Dissolution is a critical parameter of pharmaceutical dosage forms. It is well recognized that in vitro dissolution testing is done during development; screening of formulations is to ensure batch-to-batch quality control. In vitro dissolution testing is an important tool in the evaluation of the best formulation and also in the understanding of possible risks related to specific gastrointestinal environment, bioavailability and interaction with other drugs. Selection of dissolution medium is based on stability and selected on the basis of formulation used. The FDA provides guidelines for dissolution tests for oral modified release dosage forms. Very much scope for pharmaceutical person to develop suitable dissolution testing media for bioequivalence studies of newly developed formulations. The dissolution testing was performed previously in GEM formulation and AMB formulation.but dissolution study yet not had done with combination of GEM and AMB extended release formulation. Gemifloxacin is a fluoroquinolone antibiotic. Gemifloxacin chemically is 7-[(4E)-3-(aminomethyl)-4-methoxy- iminopyrrolidin--yl]--cyclopropyl-6-fluoro-4-oxo-,8- aphthyridine-3-carboxylic acid. It is used in the treatment of chronic bronchitis and mild-to moderate pneumonia and acts by inhibiting DNA synthesis through the inhibition of both DNA gyrase and topoisomerase IV enzymes as shown in figure. Fig. : Chemical structure of Gemifloxacin Ambroxol (G-CIN A) were purchased from local Mumbai retailer Instruments A double-beam UV-Visible spectrophotometer (shimadzu UV 8) with the UV probe software was used. cm quartz cells, instrument scan speed of 6 nm min-, scan range of 2-4 nm and fixed slit width of 2 nm. Stability studies In stability study nine dissolution media were selected and prepared such as distilled water,.m HCl, ph.2 KCl-HCl buffer and ph 5.8, 6.2, 6.6, 7., 7.4 and 7.8 phosphate buffers as per USP guidelines [United States Pharmacopoeia XXX, 27]. The ph of the buffers was adjusted using Elico make ph meter. Stock solutions of GEM and AMB were prepared by dissolving accurately weighed 5 mg of both drug in 5 ml of distilled water,.m HCl, ph.2 KCl-HCl buffer and ph 5.8, 6.2, 6.6, 7., 7.4 and 7.8 phosphate buffers separately to obtain µg/ml solutions. All the solutions were sonicated using ultrasonicater to dissolve the drug. From these solutions ml was pipette out into ml volumetric flask and diluted with the same solvent system up to the mark to obtain µg/ml solutions. Two sets of µg/ml solutions of GEM and AMB are prepared and stability was tested in the above prepared dissolution media at room temperature (RT) and 37 o C in an incubator (Thermo lab ) for 48 hrs separately. These samples are studied at, 24 and 48 hrs interval by using a double-beam UVvisible spectrophotometer (shimadzu UV8) connected to UV probe software. The λmax and absorbance value was measured for all the solutions and deviations in the values are recorded which indicates stability in.m HCl, 6.6 and 7 Phosphate buffer respectively. These stable dissolution medias are used for further dissolution studies of both the drugs as shown in figure 3..8.6 A bs.4 Fig. 2: Chemical structure of Ambroxol Ambroxol, chemically is 4-[[(2-amino-3,5- dibromophenyl )-methyl]-amino] cyclohexanol, an active metabolite of bromhexine, is a Mucolytic agent useful in chronic obstructive bronchitis(merck index 777 ). The main aimed of investigation is to developing and optimized the stability indicating dissolution media for conducting dissolution studies of GEM and AMB from extended release tablets as shown in figure 2. Material And Methods Procurement of drug and chemical Bulk drug of Gemifloxacin was procured from Hetero Pharmaceutical Hyderabad, India. Ambroxol was procured from Aristo Pharmaceutical Mumbai, India. Potassium dihydrogen ortho phosphate of AR grade and other chemicals of AR grade were purchased from Fischer Ltd, Mumbai. Water used was double distilled grade. Pharmaceutical Formulations of Gemifloxacin and.2 2 25 3 35 4 W avelength [nm ].8.6 A bs.4.2 2 25 3 35 4 W avelength [nm ] Fig. 3: Dissolution profile of Gemifloxacin and Ambroxol in. MHCL, 6.6, 7 phosphate buffer 2.4 Development and validation of selected dissolution media by analytical methods Calibration curve of GEM and AMB were constructed in selected stable dissolution media. orbance was 5
2.5.8.5 2 25 3 35 4.6.4.2 2 25 3 35 4 Fig.4: (a). Overlain spectra of GEM and AMB in. M HCL, 6.6, 7 phosphate buffer at time intervals of, 24 and 48 hrs and temperature 37 o C 2 2.5.5.5.5 2 25 3 35 4 2 25 3 35 4 Fig. 4: (b) Overlain spectra of GEM and AMB in ph 6.6 phosphate buffer at time intervals of, 24 and 48 hrs and temperature 37 o C determined for GEM and AMB at Selected λmax values using First order derivative spectrophotometry method. The proposed methods were validated for Linearity, LOD, LOQ, Accuracy, and Precision. The method was tested for intra-day (within a day) and inter-day (within different days) precision with %RSD value less than 2%. The recovery studies were carried out at three levels (8%, %, and 2%) by adding known amounts of GEM and AMB standard drug to drug formulation and subjecting them to the proposed UV spectrophotometric methods. 2.5 Assay of GEM and AMB in commercial tablet formulations: The estimation of drug content in commercial formulations was carried out by the developed analytical method using selected dissolution media. Contents of twenty tablets containing GEM and AMB were crushed and powdered. The powder equivalent to 32mg of GEM and 7.5 mg of AMB added into selected medium and then sonicated for 2 minutes and then adjusts the volume to ml with respective solvents and then filtered through a.45 µm Whattman filter paper. From the filtrate ml was pipette into a ml volumetric flask and then the volume was adjusted to ml with respective dissolution medium and was assayed for GEM and AMB content using selected method. The above procedure was replicates of six for same concentration range as shown in figure 4. 2.6 In-vitro dissolution testing The dissolution testing was performed on the tablet G- CIN A brand of Lupin Ltd India, employing the selected stable dissolution media. The dissolution testing was carried out in a six-stage dissolution testing apparatus (Electrolab, TDT-6L). A 9ml of the selected dissolution medium was taken in basket of dissolution apparatus assembly and dissolution test was performed using USP Type 2 dissolution apparatus (paddle type) at a temp 37oC and rotation 75 rpm. Sampling is done at time interval of 5,,3, 6, 9, 2, 5 and 8 min. 5 ml of solution was withdrawn each time from the dissolution bowl and replace with equal amount of fresh dissolution media (sink condition is maintained). All the sample solutions are analyzed by First order derivative method at selected λmax of 245 nm for GEM and 292 nm for AMB using UV-Visible spectrophotometer against blank solution containing dissolution medium after suitable dilution as shown in figure 5. 52
2 Dissolution profile of GEM % Drug release 8 6 4 2.M HCL 6.6 PHOS 7 PHOS 5 5 2 Time(minutes) Dissolution profile of AMB % Drug release 2 8 6 4 2 5 5 2 Time (minutes).m HCL 6.6 PHOS 7PHOS Fig. 5. In-vitro dissolution testing profile of GEM and AMB at different ph Result and Discussion The stability of GEM and AMB solutions was performed in nine dissolution media such as distilled water,.m HCl, ph.2 KCl-HCl buffer and ph 5.8, 6.2, 6.6, 7., 7.4 and 7.8 phosphate buffers. From Stability testing it shows that drugis stable (%CV less than 2) in ph.mhcl, ph 6.6 and ph 7. phosphate buffers at Room temp and 37 o C for a period of 48 hrs in the UV region. The λ max was found to be 245 nm and 292nm for GEM and AMB respectively, using First order derivative method for.m HCl, ph 6.2 and ph 7. phosphate buffers. The dissolution studies was carried out in selected stable dissolution medias and validated as per ICH guidelines. [,2] Validation of developed First Order derivative method according to ICH guidelines-ich Q2A; ICHQ3B [2] Linearity Linearity was found to be in the range of -5 µg/ml for GEM and 2- µg/ml for AMB in the dissolution media.m HCl, ph 6.6 and ph 7.phosphate buffers respectively. For GEM y=.3x+.8, y=.25x+.2, y=.26x+.6 and for AMB y=.2x+.2, y=.2x+., y=.x+. in the dissolution media.m HCl, ph 6.6 and ph 7. phosphate buffers respectively. Regression coefficient value was.999 for both GEM and AMB. The optical parameters and regression coefficient of proposed analytical methods in selected dissolution media were summarized in Table 3. Limit of detection (LOD) and Limit of Quantitation (LOQ) The LOD and LOQ of GEM and AMB by the proposed methods were determined by using calibration curve. LOD and LOQ values were calculated by formula 3.3 σ/s and σ/s respectively, where S is the slope of the calibration curve and σ is the standard deviation of y- intercept of regression equation. Value of LOD for GEM was.88,.26, and.76 and for AMB was.55,.22 and.3 for.m HCL, ph 6.6, and ph 7 Phosphate buffer respectively. Value of LOQ for GEM was 2.667,.8, and 2.37 and for AMB.667,.833,.99 in.m HCL, ph 6.6, and ph 7 Phosphate buffer respectively. Accuracy The accuracy was determined by standard addition method. Three different levels (8%, % and 2%) of standards were spiked to commercial tablets in triplicate. The mean of percentage recoveries and the %RSD was 53
calculated. The mean % recoveries of GEM were found to be 99.52%,.5% and.% in. M HCl, ph 6.6 and ph 7.phosphate buffers respectively (Table 5a) and for AMB were found to be 99.67%, 99.% and.66 in.m HCl, ph 6.6 and ph 7.phosphate buffers respectively. Precision The repeatability of the method was determined by assaying six standard solutions of GEM at the concentration 32µg/ml, and AMB 7.5 µg/ml respectively. The reproducibility of proposed method was determined by performing tablet assay at different time intervals within a day (Intra-day precision) and on three different days (Inter-day precision). Results of Intra-day precision and Inter-day precision is expressed in %RSD which describe in Table 4. Dissolution profile Dissolution study was done in selected dissolution media of.m HCL, ph 6.6 and ph 7 Phosphate buffer at 37 o C temperature and 75RPM, Using USP Type 2dissolution apparatus (paddle type). Amount of drug releasedis calculated by using equation for GEM as y=.3x+.8, y=.25x+.2, y=.26x+.6 and for AMB y=.2x+.2, y=.2x+., y=.x+. in the dissolution media.m HCl, ph 6.6 and ph 7. phosphate buffers respectively.percent drug released of GEM was found to be 99.95 %, 99.45 %, 99.46 % in 2 minutes and for AMB was found to be97.5 %, 96.8 %,.5 % in 5 minutes for.m HCL, ph 6.6 and ph 7 Phosphate buffer respectively. [3, 4] Conclusion From the above study it is concluded that for the first time stability indicating dissolution medias are developed for dissolution studies of GEM and AMB. New First order derivative spectroscopic method was developed and validated as per ICH guidelines for detection of drug concentration and percent drug release. The developed dissolution media could be employed as simulated gastric fluid (.M HCL) and simulated intestinal fluids (ph 6.6 and ph 7. phosphate buffers) are used for study in-vitro dissolution profiles of tablets of Gemifloxacin and Ambroxol (G-CIN A). Acknowledgement The authors are thankful to Mrs.Fatma Zakaria, Chairman, Maulana Azad Educational trust, Aurangabad and Dr. Zahid Zaheer, Principal, Y. B. Chavan college of Pharmacy, Aurangabad for providing excellent laboratory facilities. References. J. C. Wang, DNA topoisomerases Annual Review of Biochemistry. 996, 65, 635 692. 2. K. Drlica, X. Zhao, DNA Gyrase, topoisomerase IV, and the 4-quinolones, Micro. Mol. Biol. Rev. 997, 6, 377 392. 3. Suran Budavari, The Merck Index-An Encyclopedia of Chemicals, Drugs and Biologicals, 3 th edition, Published by Merck research Laboratories, 382,777 & 779. 4. The United States Pharmacopoeia XXX, The United States Pharmacopoeial Convention Inc., Rockville, MD. 27, 88-92. 5. N. Ekpe, T. Jacobsen, Drug Dev. Ind. Pharm. 999, 25(9), 57. 6. Oral Extended (Controlled) Release Dosage Forms, In Vivo Bioequivalence and In Vitro Dissolution Testing, U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER), 997. 7. A. Prakash Katakam, S. Kumari, T. Nagiat, Hwisa, B. R. Chandu, Rec. Res. Sci. Tech. 2 3(7),4-45. 8. V. Balaji, S. Poongothai, B. Madhavi, Rajasekhar Reddy, P.Y. Naidu,C. M. Karrunakaran, R. Ilavarasan, Der Pharmacia Lettre, 2, 2(6), 66-75. 9. V. Balaji, S. Poongothai, B. Madhavi, Rajasekhar Reddy, P.Y. Naidu,C. M. Karrunakaran, R. Ilavarasan, int. j. Pharm. sci. 2(2), S32-35.. M. Mothilal, R. Pushpalatha, N. Damodharan, V. Manimaran, K.S. Lakshmi, Der Pharmacia Lettre, 2, 2(3), 286-296.. Kun Nie, Li Li, Xinxia Li, Yan Zhang, Xuan Mu, and Jian Chen, Dissolution Technologies. 29, 4-7. 2. www.accessdata.fda.gov/scripts/cder/dissolution. [last accessed on-//2] 3. ICH, Q2A Text on validation of analytical procedures, International Conference on Harmonization.ICH, Q3B validation of analytical procedures: methodology, International Conference on Harmonization. 54